Histidine-trytophan-ketoglutarate cardioplegia reduces inflammatory response and serum levels of myocardial enzymes in newly developed right-thoracotomy rat model

Abstract

The objective of this research was to establish a rat model for cardiopulmonary bypass (CPB) with cardiac arrest and resuscitation that is both practical and economical and simulates clinical cardiac surgery. Concurrently, the study aimed to evaluate the myocardial protective effects conferred by histidine–tryptophan–ketoglutarate (HTK) cardioplegia. Thirty rats were randomly assigned to three groups: the histidine–tryptophan–ketoglutarate (HTK), 4:1 blood cardioplegia (BC) and del Nido cardioplegia (DN) groups. The cardiopulmonary bypass (CPB) procedure was implemented and sustained for a duration of one hour. Subsequent to the cessation of CPB, the rats were subjected to monitoring and observation for an additional two hours. Following this observation period, the heart and blood samples were procured for subsequent analysis. During CPB, the average hematocrit level was significantly below the typical physiological range (P < 0.001). Histopathological scores were notably lower in the HTK group in contrast to the BC group (P < 0.001) or the DN group (P < 0.001). At 2 h after weaning off CPB, the levels of CK and CKMB in the DN and BC groups were notably elevated compared to the HTK group (P < 0.001). The levels of IL-6 and TNF-α proteins were notably increased in all three groups (P < 0.001), with the BC and DN groups showing higher increases compared to the HTK group (P < 0.001). This compact animal model of cardiopulmonary bypass (CPB) with cardiac arrest and resuscitation might allow for both the study of myocardial ischemia-reperfusion injury as well as cardioprotective strategies. HTK cardioplegia could reduce inflammatory response and serum levels of myocardial enzymes in this newly developed right thoracotomy rat model.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-76266-4.

Introduction

In order to elucidate the pathophysiological basis and the potential causes of postoperative myocardial ischemia-reperfusion injury (MIRI) of perioperative CPB injuries and explore effective protective strategies, it was necessary to establish suitable animal models for in-depth and systematic experimental studies. Leveraging larger animal models such as pigs, dogs, and sheep could necessitate significant financial commitments as well as the requirement for skilled laboratory personnel and resources¹. While normothermic cardioplegic solutions have demonstrated substantial efficacy in inducing cardiac arrest in isolated heart models, these models fail to accurately replicate the complexities inherent in cardiac surgery. In recent years, numerous research articles utilizing rat models with cardiopulmonary bypass (CPB) have been published, contributing significantly to advancements in animal testing. Nevertheless, there remains a paucity of research specifically focusing on the resuscitation of cardiac arrest via right thoracotomy—an essential procedure for studying organ damage associated with cardiac surgery. Some studies have utilized balloon occlusion of the aorta to induce cardiac arrest; however, this method requires specialized expertise and considerable financial investment². The objective of our research is to develop a rat model of cardiac arrest induced by myocardial cardioplegia through a right thoracotomy.

Optimal cardioplegic formulations ought to induce swift diastolic cessation, mitigate ischemic injury within the myocardial tissues to the greatest extent achievable, offer resistance against reperfusion harm, facilitate expedient rejuvenation of cardiac functions post-operatively, and exhibit negligible toxicity toward extracardiac systems³. Buckberg’s solution, prevalent in adult cardiac surgery, employed a predominantly extracellular approach with a 4:1 blood-to-crystalloid mixture; yet this approach necessitated repetitive administrations at intervals of thirty minutes intraoperatively. Blood intrinsically possessed buffer capacity that might curtail myocardial swelling, alongside its function of oxygen transport, enabling rapid induction of arrest under oxygenated conditions. Conversely, the HTK solution—a sodium-deficient intracellular-type cardioplegic medium—elicited functionality through the reduction of extracellular sodium levels, and had garnered clinical recognition for efficacious organ preservation⁴. Its broad adoption rested on its single-administration protocol, ensuring extended myocardial safeguarding exceeding two hours of cardiac asystole. The DN plan outlined a 4:1 ratio of crystalloid to whole blood, reducing calcium overload during ischemia reperfusion, mainly due to the active ingredients in lidocaine and magnesium. The selection of cardioplegia types was diverse and was tailored according to established clinical protocols. Despite extensive research, a definitive consensus on cardioplegic application remained elusive.

CPB led to the release of various inflammatory factors triggered by factors such as cold exposure, surgery, anesthesia, ischemia-reperfusion injury, non-physiological perfusion, endotoxin release, coagulation abnormalities, and heparin/fish protein reaction. These factors included IL-6, IL-8, and TNF-α. Ischemia-reperfusion injury is the primary cause of the disease’s pathological changes during CPB, leading to a shift in cardiomyocyte metabolism from aerobic to anaerobic, ultimately triggering systemic inflammatory response syndrome (SIRS)⁵. Considering these previous factors, we aimed to evaluate the effectiveness of HTK, BC, and DN solutions through various parameters including light microscopy results, electron microscopic observations, serum levels of myocardial enzymes, and the expression patterns of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) using the rat model described in this study.

Results

Hemodynamic parameters

No significant variation was observed in the hemodynamics among the three groups throughout the perioperative period. The average blood pressure and heart rate declined when the CPB started. After the 2-hour post-CPB evaluation, the physiological measurements, such as heart rate and average blood pressure, reverted back to their initial levels (Table 1). The flow rate was kept between 100 and 150 mL/kg/min during the management of CPB.

Table 1.

Hemodynamic Parameters of CPB during perioperative period.

	T1	T2	T3	T4	T5	T6	T7	T8	T9
Weight (g)
HTK	446.6 ± 14.17
BC	445.8 ± 19.54
DN	448.4 ± 10.83
CPB flow (ml/kg/min)
HTK	–	67.20 ± 5.63	116.00 ± 4.18	155.00 ± 6.12	164.20 ± 2.39	166.00 ± 4.18	170.00 ± 11.73	–	–
BC	–	61.00 ± 4.18	113.00 ± 7.58	150.00 ± 7.91	155.00 ± 6.12	162.00 ± 2.74	165.00 ± 3.54	–	–
DN	–	61.60 ± 2.88	114.40 ± 3.78	151.00 ± 4.18	157.00 ± 4.47	162.00 ± 2.74	164.00 ± 4.18	–	–
MAP (mmHg)
HTK	104.60 ± 4.56	87.00 ± 4.30	87.20 ± 1.30	55.40 ± 2.97	53.20 ± 3.03	57.60 ± 2.07	76.40 ± 1.52	96.00 ± 1.58	100.60 ± 1.52
BC	109.40 ± 4.39	87.20 ± 1.30	85.00 ± 2.24	56.20 ± 5.63	55.60 ± 2.70	61.00 ± 2.74	77.80 ± 3.19	101.00 ± 4.06	99.20 ± 6.80
DN	106.60 ± 2.70	85.40 ± 1.82	82.40 ± 1.67	59.60 ± 3.78	57.20 ± 3.11	61.20 ± 2.77	77.40 ± 2.51	100.80 ± 2.17	103.40 ± 1.82
PaCO2 (mmHg)
HTK	44.00 ± 1.58	44.60 ± 1.14	43.00 ± 1.58	43.20 ± 1.92	43.60 ± 1.52	43.00 ± 1.58	43.80 ± 0.84	43.80 ± 1.92	42.60 ± 1.95
BC	43.40 ± 2.51	43.40 ± 1.82	44.40 ± 1.52	43.60 ± 2.41	43.60 ± 2.07	44.00 ± 2.12	44.40 ± 1.82	41.60 ± 1.14	42.00 ± 0.71
DN	43.00 ± 1.58	41.40 ± 1.34	43.80 ± 0.84	43.00 ± 1.58	43.40 ± 0.89	43.80 ± 1.10	43.80 ± 1.30	41.60 ± 1.14	42.00 ± 0.71
PaO2 (mmHg)
HTK	104.20 ± 3.19	106.00 ± 4.30	209.80 ± 7.76	216.00 ± 9.57	202.40 ± 9.24	204.80 ± 7.98	196.00 ± 9.62	106.60 ± 4.22	99.60 ± 3.91
BC	108.60 ± 3.05	105.20 ± 3.96	211.60 ± 4.77	219.20 ± 1.10	211.00 ± 6.52	202.60 ± 3.71	205.80 ± 12.60	104.80 ± 2.39	99.00 ± 4.64
DN	105.80 ± 2.49	108.60 ± 2.97	210.80 ± 7.29	214.40 ± 6.07	209.20 ± 2.59	209.00 ± 6.52	206.40 ± 10.60	105.80 ± 3.77	104.80 ± 3.27
HR (times/min)
HTK	282.00 ± 5.15	259.20 ± 6.83	241.20 ± 7.63	–	–	–	149.80 ± 6.57	229.00 ± 11.20	235.80 ± 9.63
BC	275.60 ± 6.88	257.80 ± 4.02	246.40 ± 3.85	–	–	–	152.20 ± 6.06	226.20 ± 7.29	230.80 ± 5.76
DN	276.40 ± 7.02	261.40 ± 5.37	245.80 ± 7.56	–	–	–	151.40 ± 6.80	230.00 ± 10.91	235.00 ± 7.81
T (°C, rectal)
HTK	35.88 ± 0.25	35.56 ± 0.19	33.58 ± 0.26	32.02 ± 0.19	31.72 ± 0.28	33.32 ± 0.24	34.20 ± 0.35	35.86 ± 0.11	36.12 ± 0.13
BC	35.78 ± 0.28	35.52 ± 0.31	33.78 ± 0.37	32.16 ± 0.22	31.78 ± 0.22	33.28 ± 0.47	34.38 ± 0.16	35.78 ± 0.20	36.04 ± 0.19
DN	35.80 ± 0.27	35.52 ± 0.31	33.82 ± 0.37	32.12 ± 0.15	31.76 ± 0.24	33.08 ± 0.46	34.34 ± 0.15	36.04 ± 0.18	36.18 ± 0.11

CPB, cardiopulmonary bypass; MAP, mean arterial pressure; PaCO₂, partial pressure of carbon dioxide; PaO₂, partial pressure of oxygen; HR, heart rate; T, temperature. Significance levels were denoted as ^*P < 0.05, ^**P < 0.01, and^***P < 0.001 when compared to HTK, and as ^#P < 0.05 and ^##P < 0.01 when compared to BC.

Evaluation of blood gas levels, electrolytes, and the balance of acid-base in the body

During the perioperative period, Table 2 displayed the electrolyte levels, acid-base balance, and metabolic parameters of CPB for three groups. The average hematocrit level decreased significantly during CPB compared to the initial physiological values, mainly due to hemodilution caused by priming. Nevertheless, following discontinuation of CPB, the hematocrit values slowly reverted back to their original levels. At 10 min after cardiac arrest, the levels of sodium ions in the HTK group were significantly lower compared to the BC group (129.7 ± 1.2 VS 129.7 ± 1.0, P = 0.004) and the DN group (126.7 ± 1.2 VS 130.4 ± 1.2, P = 0.001). Similarly, the calcium ion levels in the HTK group were also lower than the BC group (1.28 ± 0.11 VS 1.34 ± 0.01, P = 0.000) and the DN group (1.28 ± 0.11 VS 1.33 ± 0.02, P = 0.001). At 20 min post arrest, the hematocrit levels of the HTK group were significantly lower compared to both the BC group (21.0 ± 0.0 vs. 22.6 ± 0.5, P = 0.000) and the DN group (21.0 ± 0.0 vs. 22.6 ± 0.5, P = 0.002).The hematocrit levels of the HTK and DN groups were found to be lower compared to the BC group (26.6 ± 0.9 vs. 28.4 ± 0.5, p = 0.024; 25.6 ± 1.1 vs. 28.4 ± 0.5, p = 0.001) upon discontinuation of CPB. Five-line charts were used to illustrate the changes in important physiological factors such as sodium ion, potassium ion, calcium ion, hematocrit, and lactate throughout the arrest-resuscitation process. (Fig. 1(A)-(E))

Table 2.

Blood gas, electrolyte levels, acid-base balance, and metabolic parameters of CPB throughout the perioperative period.

	T1	T2	T3	T4	T5	T6	T7	T8	T9
Glucose (mmol/L)
HTK	8.10 ± 0.25	9.60 ± 0.35	13.28 ± 0.49	16.52 ± 0.56	15.64 ± 0.43	14.86 ± 0.55	13.48 ± 0.48	12.04 ± 0.52	11.74 ± 0.48
BC	8.04 ± 0.47	9.50 ± 0.40	12.94 ± 0.61	15.80 ± 0.60	14.96 ± 0.47	14.08 ± 0.58	12.94 ± 0.48	11.26 ± 0.68	11.04 ± 0.73
DN	8.12 ± 0.16	9.64 ± 0.38	13.34 ± 0.42	16.16 ± 1.05	15.32 ± 0.48	14.38 ± 0.73	13.44 ± 0.53	11.62 ± 0.57	11.32 ± 0.58
pH (arterial)
HTK	7.38 ± 0.05	7.36 ± 0.03	7.39 ± 0.05	7.39 ± 0.03	7.27 ± 0.05	7.33 ± 0.02	7.38 ± 0.03	7.40 ± 0.02	7.39 ± 0.03
BC	7.43 ± 0.05	7.40 ± 0.04	7.40 ± 0.03	7.37 ± 0.01	7.26 ± 0.06	7.34 ± 0.01	7.41 ± 0.03	7.43 ± 0.06	7.40 ± 0.04
DN	7.43 ± 0.03	7.40 ± 0.03	7.39 ± 0.03	7.38 ± 0.01	7.27 ± 0.04	7.33 ± 0.01	7.40 ± 0.02	7.40 ± 0.02	7.39 ± 0.03
HCO3− (mmol/L)
HTK	23.06 ± 0.67	21.96 ± 0.59	20.96 ± 0.74	20.72 ± 0.79	20.50 ± 0.86	20.20 ± 0.72	20.82 ± 0.43	21.44 ± 0.29	21.70 ± 0.31
BC	22.74 ± 0.78	22.08 ± 0.61	21.26 ± 0.57	20.90 ± 0.60	20.62 ± 0.73	20.20 ± 0.59	20.86 ± 0.37	21.44 ± 0.29	21.84 ± 0.42
DN	22.98 ± 0.36	22.38 ± 0.48	21.70 ± 0.16	21.36 ± 0.17	21.08 ± 0.19	20.54 ± 0.72	21.08 ± 0.41	21.50 ± 0.33	21.72 ± 0.34
BE (mmol/L)
HTK	− 3.94 ± 0.34	− 4.34 ± 0.22	− 4.38 ± 0.31	− 5.26 ± 0.44	− 5.88 ± 0.54	− 5.82 ± 0.55	− 5.14 ± 0.29	− 4.06 ± 0.21	− 4.26 ± 0.15
BC	− 3.74 ± 0.11	− 4.28 ± 0.29	− 4.32 ± 0.08	− 5.32 ± 0.13	− 6.30 ± 0.10	− 5.64 ± 0.35	− 4.98 ± 0.24	− 4.16 ± 0.32	− 4.28 ± 0.58
DN	− 4.06 ± 0.21	− 4.4 ± 0.21	− 4.3 ± 0.29	− 5.46 ± 0.66	− 5.94 ± 0.50	− 5.60 ± 0.52	− 5.14 ± 0.34	− 4.16 ± 0.17	− 4.36 ± 0.19
K+(mmol/L)
HTK	4.46 ± 0.23	4.26 ± 0.23	4.28 ± 0.13	5.72 ± 0.42	5.58 ± 0.41	5.30 ± 0.29	4.90 ± 0.29	4.62 ± 0.26	4.44 ± 0.23
BC	4.44 ± 0.23	4.30 ± 0.17	4.26 ± 0.15	6.22 ± 0.19	6.18 ± 0.36	5.34 ± 0.21	4.82 ± 0.24	4.52 ± 0.24	4.34 ± 0.21
DN	4.50 ± 0.28	4.38 ± 0.23	4.28 ± 0.16	5.76 ± 0.38	6.48 ± 0.15	5.78 ± 0.08	4.70 ± 0.16	4.46 ± 0.11	4.28 ± 0.08
Hct (%)
HTK	41.20 ± 1.64	39.40 ± 1.14	24.60 ± 0.55	23.80 ± 0.84	22.20 ± 0.45	21.00 ± 0.00	23.40 ± 0.55	26.60 ± 0.89##	34.40 ± 1.14
BC	41.80 ± 1.30	39.80 ± 1.30	25.20 ± 0.84	24.20 ± 0.84	23.00 ± 0.71	22.60 ± 0.55***	24.40 ± 0.55	28.40 ± 0.55	34.60 ± 0.71
DN	41.40 ± 1.34	40.20 ± 0.84	24.80 ± 0.84	23.80 ± 0.84	22.00 ± 0.71	22.20 ± 0.45**	24.20 ± 0.84	25.60 ± 1.14##	34.40 ± 1.14
Lac (mmol/L)
HTK	0.68 ± 0.08	0.52 ± 0.08	1.64 ± 0.11	1.96 ± 0.11	2.20 ± 0.12	2.48 ± 0.08	2.90 ± 0.16	3.24 ± 0.21	3.80 ± 0.42
BC	0.72 ± 0.18	0.52 ± 0.13	1.52 ± 0.24	1.82 ± 0.19	2.08 ± 0.23	2.38 ± 0.22	2.64 ± 0.18	3.04 ± 3.04	3.62 ± 0.49
DN	0.72 ± 0.13	0.54 ± 0.11	1.56 ± 0.11	1.88 ± 0.13	2.14 ± 0.05	2.42 ± 0.08	2.88 ± 0.19	3.24 ± 0.21	3.78 ± 0.44
Na+(mmol/L)
HTK	141.88 ± 3.66	139.32 ± 3.62	136.80 ± 3.03	133.04 ± 0.92	126.74 ± 1.17	129.58 ± 2.11	131.88 ± 1.61	134.92 ± 1.10	135.24 ± 0.92
BC	142.06 ± 3.09	139.16 ± 1.94	136.12 ± 1.27	133.68 ± 2.34	129.72 ± 0.98**	130.58 ± 0.91	131.74 ± 2.28	134.46 ± 2.36	134.80 ± 2.32
DN	142.08 ± 4.40	137.52 ± 3.01	135.00 ± 0.94	132.80 ± 0.51	130.38 ± 1.24**	127.12 ± 3.79	130.28 ± 2.38	133.54 ± 1.81	133.98 ± 1.80
Ca2+(mmol/L)
HTK	1.482 ± 0.03	1.438 ± 0.01	1.41 ± 0.01	1.38 ± 0.01	1.28 ± 0.01	1.31 ± 0.01	1.34 ± 0.01	1.38 ± 0.01	1.42 ± 0.02
BC	1.482 ± 0.03	1.438 ± 0.02	1.40 ± 0.02	1.37 ± 0.01	1.34 ± 0.01***	1.33 ± 0.01	1.34 ± 0.01	1.38 ± 0.01	1.44 ± 0.01
DN	1.482 ± 0.02	1.448 ± 0.02	1.42 ± 0.01	1.38 ± 0.01	1.33 ± 0.02**	1.31 ± 0.02	1.34 ± 0.01	1.38 ± 0.01	1.43 ± 0.02

CPB, cardiopulmonary bypass; Lac, lactate; Hct, hematocrit; HCO₃⁻, Standard bicarbonate; BE, Base excess; PH, hydrogen ion concentration. Significance levels were denoted as ^*P < 0.05, ^**P < 0.01, and^***P < 0.001 when compared to HTK, and as ^#P < 0.05 and ^##P < 0.01 when compared to BC.

Fig. 1.

The fluctuation pattern of essential physiological electrolyte levels throughout the arrest and resuscitation procedure. Significance levels were denoted as^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 when compared to HTK, and as ^#P < 0.05 and ^##P < 0.01 when compared to BC.

Pathological analysis of myocardial tissue

We conducted H&E staining on cardiomyocytes to evaluate the overall structure of the myocardium. In HTK group, the myocardial tissue structure appeared predominantly normal, with well-organized myocardial fibers. Mild interstitial edema was observed in certain regions, accompanied by mild infiltration of inflammatory cells. No evidence of ischemic necrosis in myocardial cells was detected. In BC group, the myocardial tissue structure showed a mostly normal pattern, although there was slight disarray in the arrangement of myocardial fibers. Some areas exhibited mild interstitial edema and moderate inflammatory reaction, occasionally accompanied by apoptotic and necrotic cells. In DN group, the myocardial tissue structure displayed a generally normal appearance, albeit with slightly disordered arrangement of myocardial fibers. Mild edema was present within the myocardium along with moderate infiltration of inflammatory cells. A few apoptotic and necrotic cells were also observed. Histopathological scores were 4.49 ± 0.18 in the HTK group, 5.26 ± 0.26 in the BC group, and 5.43 ± 0.20 in the DN group. Histopathological scores were notably reduced in the HTK group in comparison to both the BC and DN groups, with statistical significance (P < 0.001). The BC and DN groups did not show a notable distinction (P = 0.091). (Fig. 2 (A)-(D))

Fig. 2.

The results of light microscope of three groups (X20). Significance levels were denoted as ^###P < 0.001 when compared to HTK.

Electron microscopic structure of myocardium

The mitochondrial structure was basically normal of three groups. In HTK group, the mitochondrial structure appeared predominantly normal, with a loss of mitochondrial particles and some swollen mitochondria. The substrate showed a more uniform distribution, while the ridge structure remained largely intact. In BC group, the mitochondrial structure was mostly normal, with a few lost mitochondrial particles and visible changes in the ridge structure. In DN group, the mitochondrial structure was essentially normal, although there was partial loss of mitochondrial particles and slight swelling in some mitochondria. The matrix exhibited a more uniform composition, along with minor alterations in the ridge structure. The score for mitochondrial injury was 1.85 ± 0.23 in the HTK group, 1.84 ± 0.30 in the BC group, and 1.82 ± 0.28 in the DN group. Nevertheless, there were no notable distinctions noted when comparing the HTK group to both BC and DN groups (p = 0.806), as well as between BC and DN groups in terms of their mitochondrial damage levels (p = 0.870). (Fig. 3(A)-(D))

Fig. 3.

The results of electron microscope of three groups (X10000).

Serum levels of myocardial enzymes creatine kinase (CK), creatine kinase MB (CKMB), lactate dehydrogenase (LDH), and lactate dehydrogenase 1 (LDH1)

No notable variances were observed in the levels of serum CK, CKMB, LDH, and LDH1 before CPB among the three groups (P = 0.991, P = 0.922, P = 0.866, P = 0.882). Levels of Serum CK, CKMB, and LDH were notably elevated at 2 h after weaning off CPB compared to before CPB (P < 0.001 for each). At 2 h after weaning off CPB, the levels of CK and CKMB in the BC and DN groups were notably elevated compared to the HTK group (all P < 0.001). However, there was no statistical difference between BC and DN groups (P = 0.815, P = 0.816, respectively). No notable variances were observed in serum LDH and LDH1 levels across the three groups (P = 0.540, P = 0.291, respectively) (Table 3; Fig. 4).

Table 3.

Changes of myocardial enzyme spectra in the three groups.

	HTK	BC	DN	P b
CK(U/L)
T1	260.9 ± 47.7	262.6 ± 41.5	261.2 ± 42.1	0.991
T9	736.7 ± 42.3	968.4 ± 63.7	962.5 ± 59.1	< 0.001
Pa	< 0.001	< 0.001	< 0.001
CKMB(U/L)
T1	190.5 ± 19.6	188.5 ± 21.2	187.0 ± 16.5	0.922
T9	561.2 ± 53.8	762.5 ± 44.6	767.6 ± 44.1	< 0.001
Pa	< 0.001	< 0.001	< 0.001
LDH(U/L)
T1	445.1 ± 42.3	455.1 ± 45.8	454.5 ± 51.7	0.866
T9	843.2 ± 30.3	859.3 ± 33.8	855.2 ± 35.4	0.540
Pa	< 0.001	< 0.001	< 0.001
LDH1(U/L)
T1	8.4 ± 1.4	8.1 ± 1.3	8.3 ± 1.2	0.882
T9	10.2 ± 3.7	8.6 ± 1.6	8.7 ± 1.4	0.291
Pa	0.184	0.530	0.579

T1, Pre-CPB; T9, 2 h post CPB; CK, creatine kinase, CKMB, creatine kinase MB; LDH, lactate dehydrogenase; LDH1, lactate dehydrogenase 1; BC, 4:1 blood cardioplegia; DN, del Nido; HTK, histidine–tryptophan–ketoglutarate; P^a, compared to T1; P^b, compared to HTK.

Fig. 4.

Histogram of serum CK, CKMB, LDH and LDH1 concentration. ^***represents P < 0.001. T1, Pre-CPB; T9, 2 h post CPB; CK, creatine kinase; CKMB, creatine kinase MB; LDH, lactate dehydrogenase; LDH1, lactate dehydrogenase 1; BC, 4:1 blood cardioplegia; DN, del Nido; HTK, histidine–tryptophan–ketoglutarate.

Protein expression levels of IL-6 and TNF-α

Protein expression of IL-6 and TNF-α in the myocardium was determined using Western blot analysis. IL-6 and TNF-α protein expression was markedly increased in all three groups, with BC and DN groups showing higher upregulation compared to the HTK group (P < 0.001) Table 4; Fig. 5(A)-(D)).

Table 4.

Protein expression of IL-6 and TNF-α in myocardium.

	Ctrl	HTK	BC	DN
IL-6	0.31 ± 0.02	0.64 ± 0.02***	0.85 ± 0.03***###	0.85 ± 0.03***###
TNF-α	0.41 ± 0.02	0.59 ± 0.04***	1.00 ± 0.05***###	1.03 ± 0.08***###

Protein expression of IL-6 and TNF-α in myocardium. Ctrl, Control group, normal rats; HTK, histidine–tryptophan–ketoglutarate; BC, 4:1 blood cardioplegia; DN, del Nido. IL-6, interleukin-6, TNF-α, tumor necrosis factor-α; ^*represents comparison to Ctrl group, ^*** represents P < 0.001; ^# represents comparison to HTK group, ^### represents P < 0.001.

Fig. 5.

The protein expression of IL-6 and TNF-α in myocardium. Ctrl, Control group (normal rats); HTK, histidine–tryptophan–ketoglutarate; BC, 4:1 blood cardioplegia; DN, del Nido; IL-6, interleukin-6, TNF-α, tumor necrosis factor-α; ^*represents comparison to Ctrl group, ^*** represents P < 0.001; ^# represents comparison to HTK group, ^### represents P < 0.001.

Discussion

Research in open heart surgery has primarily focused on protecting myocardium, but there is still debate over the best strategy for cardiac protection and which cardioplegia is most effective in preserving myocardial function after MIRI. Cardiac surgery inevitably leads to MIRI, potentially causing postoperative myocardial dysfunction, such as pulmonary dysfunction, atrial fibrillation, and renal failure⁶. Currently, there are two primary categories of cardiac arrest solutions: one containing high levels of potassium, magnesium, and bicarbonate in the extracellular fluid, and the other containing electrolyte components in the intracellular fluid^4,7. The use of cardioplegia not only induces cardiac arrest but also reduces ischemia/reperfusion damage and improves immediate or long-lasting results^8–12.Understanding of the intricate factors contributing to myocardial injury remains limited, with perioperative MIRI identified as the primary reason^13–16. Each of the three cardioplegia solutions has its own advantages, but the effects of myocardial protection are still unclear. Therefore, we compared the myocardial protection effects of three kinds of cardioplegias from different aspects.

Most of the existing literatures were partial cardiopulmonary bypass models established at room temperature or low temperature, the disadvantage of which was that it mainly simulated parallel circulation and did not open chest and might could not reflect the pathological changes of the heart and lungs during most cardiac operations^2,17–20. The cardiac arrest and resuscitation rat model through a right thoracotomy established in this experiment simulated the clinical cardiac surgery, which might be an experimental tool for studying the mechanism of cardiopulmonary injury and evaluating various cardiopulmonary protection measures. The innovation points and advantages of this model:¹ This model simulated the procedure of clinical heart surgery including cardiac arrest and resuscitation². The entire CPB circuit had a volume of under 8 mL and does not require blood precharge. A separate pump aided in drainage, transitioning from gravity drainage to pump-assisted drainage, with the pump maintaining a flow rate of 100–150 ml/kg/min³. At the onset of the experiment, we performed a midline sternotomy and inserted an aortic cannula for cardioplegia injection; however, this approach posed potential risks of trauma and bleeding, which were unfavorable for sternal closure (Fig. 6). Therefore, we further improved and optimized the model. In order to simulate the clinical minimally invasive surgery, the right small incision thoracotomy was used to expose the ascending aorta. A 20-gauge catheter, functioning as a tube for delivering cardioplegia, was inserted in a retrograde manner into the ascending aorta through the right common carotid artery. This method effectively prevented bleeding that may occur from the infusion of cardioplegic fluid into the aortic arch and maintained more stable hemodynamics and meanwhile reduced the trauma (Fig. 7).

Fig. 6.

Cardiopulmonary bypass with cardiac arrest and resuscitation in a median thoracotomy rat experiment. O₂, oxygen; EJV, external jugular vein; CA, caudal artery; AAo, ascending aorta; IBP, invasive blood pressure.

Fig.7.

Cardiopulmonary bypass with cardiac arrest and resuscitation in a right thoracotomy rat experiment. O₂, oxygen; EJV, external jugular vein; CA, caudal artery; AAo, ascending aorta; IBP, invasive blood pressure.

The study utilized myocardial enzymes CK, CK-MB, LDH, and LDH1 as indicators of myocardial damage, with the serum levels of these enzymes at 2 h after weaning off CPB serving as measures for the clinical trial. After the operation, the findings indicated a notable rise in CK, CK-MB, and LDH levels 2 h after weaning off CPB, indicating the presence of myocardial ischemia-reperfusion injury across all three groups. Levels of CK and CK-MB were notably reduced in the HTK group compared to the BC and DN groups 2 h after weaning off CPB, suggesting that myocardial ischemia-reperfusion injury was less pronounced in the HTK group. The salutary effects of HTK solution can be ascribed to the buffering capacity of histidine that potentiates the enzymatic processes of anaerobic glycolysis. Moreover, ketoglutarate, which is a precursor of nicotinamide adenine dinucleotide (NAD), plays a crucial role as an intermediate in the tricarboxylic acid cycle. The inclusion of mannitol contributes to the diminution of cellular edema. Furthermore, empirical evidence has corroborated the efficacy of HTK in maintaining the integrity of the coronary endothelium, thereby facilitating enhanced cardiac recuperation²¹.

SIRS often occurs as a secondary complication following cardiopulmonary bypass (CPB). Nevertheless, in excess of one-quarter of such cases escalate into conditions with graver manifestations, thereby amplifying postoperative morbidity and, in the most severe instances, precipitating mortality²². We evaluated the overall physical appearance and inflammation level of myocardium by conducting hematoxylin and eosin (H&E) staining on myocardial cells. To obtain more evidence, we extracted interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) from cardiac tissue samples to perform Western blot tests. IL-6, a pro-inflammatory cytokine, is released by lymphocytes, fibroblasts, macrophages, and endothelial cells, and it plays a crucial role in controlling inflammation and immune responses. It was found that the IL-6 content in the body increased significantly when the CPB began, and reached a peak after CPB²³. Several research studies have shown that the rise in IL-6 levels following cardiac bypass surgery can lead to a higher likelihood of various postoperative complications, including acute respiratory distress syndrome, renal damage, heart muscle ischemia, and reduced cardiac output. At the same time, IL-6 can also be used as a marker of disease severity, persistent inflammatory processes, or cardiac insufficiency^24,25. TNF-α is a frequently occurring proinflammatory cytokine that triggers the acute phase response, impacting cell growth, tumor development, stress reactions, and immune system reactions. TNF-α is crucial in the initiation and progression of inflammatory reactions, and is associated with the development of various complications following CPB²⁶. Therefore, IL-6 and TNF-α were selected as indicators of inflammatory factors in this study. Light microscopy and western blot analysis revealed that the inflammation of cardiomyocytes was less severe in the HTK group compared to the BC and DN groups, with no significant difference observed between the BC and DN groups.IL-6 and TNF-α protein levels were lower in HTK compared to BC and DN. These results indicated that HTK was superior to the other two groups in alleviating pathological damage and myocardial inflammatory response.

The primary constraint in our research was the small number of participants, in addition to the restricted observational measures and the absence of other biomarkers like brain natriuretic peptide (BNP). Furthermore, this study recognized limitations due to the lack of standardized experimental settings and ongoing monitoring, which prevented the long-term observation of rat survival. These limitations were built into the typical approaches used in experiments involving rat heart surgeries. Rats were deemed viable for long-term survival if their respiration, blood pressure, and heart rate remained stable 2 h post-CPB²⁷. Consequently, this study opted to evaluate rat survival at 2-hour postoperative mark. Nonetheless, the creation of enduring survival models in rodents demands meticulous oversight of anesthesia levels, continuous maintenance of cardiovascular and pulmonary functions during the procedure, and strict adherence to sterile surgical protocols. These elements will constitute the principal focus of our forthcoming research aimed at refining this model. Due to our exclusive concentration on enhancing cardiopulmonary bypass (CPB) techniques involving cardiac arrest and resuscitation, a systematic investigation into definitive outcomes and the development of long-term survival models has not been conducted. We focused on the comparison of three different cardioplegias without a blank control and intended to set a control group in a subsequent study to make the whole experiment more rigorous.

This compact animal model of cardiopulmonary bypass (CPB) with cardiac arrest and resuscitation might allow for both the study of myocardial ischemia-reperfusion injury as well as cardioprotective strategies. HTK cardioplegia could reduce inflammatory response and serum levels of myocardial enzymes in this newly developed right thoracotomy rat model.

Materials

Study approval

The protocol for the animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University with the reference number of IACUC2004003. The procedures were performed in accordance with the approved guidelines. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.

Animal preparation

Male Sprague-Dawley rats aged 14–16 weeks and weighing 350–450 g were housed in a pathogen-free environment with humidity between 45% and 55% and temperature maintained at 23–25 °C. They were kept on a 12-hour light/dark cycle and provided with unlimited food and water for a week. All animals were purchased from the Animal Experimental Center of Nanjing Medical University (Jiangsu, China). All animals were euthanized after the experiment.

Anesthesia

The test rats were anesthetized using 2–2.5% isoflurane with a FiO₂ of 0.5 in a plastic induction box. After performing orotracheal intubation using a 14G cannula (Insyte BD Medical, Sandy, UT), the rats were placed on mechanical ventilation provided by Teli Anesthesia Breathing Equipment Company, Jiangxi, China. The ventilatory parameters were set to include an FiO₂ of 0.21, a tidal volume adjusted for body weight (8–10 ml/kg), a positive end-expiratory pressure (PEEP) of 5 cm H₂O, and a respiratory rate ranging from 60 to 80 breaths per minute to maintain normal levels of oxygen and carbon dioxide. During the ensuing surgical preparation, anesthesia was continuously administered via infusion of midazolam and fentanyl, at dosages of 7 mg/kg/h and 70 µg/kg/h respectively. These anesthetic agents were procured from Hameln Pharma Plus in Germany and Rotexmedica in Germany. Venous access was established in the right femoral vein using a 24-gauge catheter (procured from BD Medical, Sandy, UT, USA) to enable the intravenous administration of pharmacological agents and fluid replenishment. At the same time, a catheter of the same size was placed in the right femoral artery to allow for careful monitoring of arterial pressure and to collect samples for blood gas analysis. They were positioned dorsally with the inclusion of a rectal probe designed for thermocouple temperature measurements. To ensure thermal homeostasis, a controlled heating platform sustained their core temperatures between 37.5 and 38.5 Celsius. Electrocardiogram (EKG) electrodes were placed on the anterior and left hind paws of rats.

CPB circuit Preparation

The compact CPB system consisted of a venous reservoir, a sterile silicone tubing circuit with a 4 mm inner diameter for the venous line and 1.6 mm for the arterial line, a custom membranous oxygenator for small animals, and a small roller pump primed with 7 mL of saline and 1 mL (1000 IU) of heparin. The starting blend, made up of 6% HES 130/0.4, NaCl (Chiatai TianQing, Nanjing, China), 5% NaHCO₃, and 20% human serum albumin (diluted to 5%), had a volume less than 8 ml. (Fig. 8).

Fig. 8.

Cardiopulmonary bypass with catheter and pipe connection. Peristaltic pump, 24-gauge catheter for measuring blood pressure, 22-gauge catheter for infusing arteries, custom-made 18-gauge cannula for draining veins, and tubes.

Establishment of CPB

A 22-gauge catheter (BD Medical, Sandy, UT) was inserted into the caudal artery serving as the CPB inflow cannula. Following the insertion of the arterial inflow cannula, 500 IU/kg of heparin sodium was given. A modified 18-gauge catheter (BD Medical, Sandy, UT) with five side-holes was used to achieve CPB outflow, being inserted into the right atrium through the right jugular vein. A 20-gauge catheter, functioning as a tube for cardioplegia perfusion, was inserted in a retrograde manner into the ascending aorta through the right common carotid artery. (Fig. 7)

The management of CPB

Following the insertion of arterial and venous tubes and connecting them, we turned on the oxygen before starting the CPB. The parameters were adjusted as follows: oxygen flow was 0.8 L/min, FiO₂ 100%, the respiratory rate was down to 30 per minute, the pressure of arterial oxygen was 200–400 mmHg. Prior to initiating CPB, the temperature setting for heating was raised to a maximum of 42℃ in order to prevent a sudden decrease in body temperature when CPB started. CPB was started with a flow rate of 100–150 ml/kg/min and rapid cooling to a rectal temperature of 32 °C was promptly carried out. Moreover, the MAP was above 60 mmHg and the Hct was above 21% during the CPB.

The management of surgical procedure

Surgical procedure management included cardioplegic arrest and resuscitation following a right thoracotomy at the 3–4 intercostal space. Tourniquets were utilized to clamp the aortic arch and brachiocephalic trunk. Following 15 min of cardiopulmonary bypass, the aortic cross-clamp was placed as 0.5 ml of cardioplegia was quickly delivered through a cardioplegia perfusion tube using an infusion pump to induce diastolic cardiac arrest. If automatic heart resuscitated, 0.5 ml cardioplegic solution was administered again. Table 5 displayed the specific ingredients of the cardioplegia solutions. The specific infusion methods and dosages of the three groups of cardioplegia were shown in Table 6. Following 30 min of myocardial ischemia, the aortic cross-clamp was removed and the heart underwent reperfusion while being warmed to a rectal temperature of 36 °C.At the same time, calcium chloride (Shen Wei Pharmaceutical Co.Ltd.10 mL/g 0.3mL, Sichuan, China) and epinephrine (Sanchine Pharmaceutical Co. Ltd. 1 mL/1 mg, Heilongjiang, China, 5ug) were given one after the other to help revive the cardiac muscle. Following a 15-minute reperfusion period, the animals were removed from cardiopulmonary bypass. Following CPB, the leftover priming solution was concentrated using the Heraeus 600i centrifuge to increase hematocrit levels before being injected into the rat. Subsequently, the cannulas were extracted, and the incisions were stitched. After monitoring post-CPB for a duration of 2 h, all the animals were subsequently euthanized. The heart and blood were sampled after euthanized for further evaluation.

Table 5.

Composition and concentration of the three cardioplegias.

	Ratio (Blood: Crystalloid)	Con (mM) Type	K+	Ca²+	Na+	Mg²+	Mannitol	Lidocaine	Histidine
Crystalloid cardioplegia	–	HTK	9	0.015	15	4	18	–	180
Blood cardioplegia	4:1	BC	20	1.3	147.9	7	–	0.085	–
	1:4	DN	24	0.4	150	6	2.6(g/L)	140(mg/L)	–

Table 6.

The specific infusion methods and dosages of cardioplegias.

	HTK	BC	DN
Perfusion temperature	4℃~6℃	4℃~6℃	4℃~6℃
Perfusion pressure(mmHg)	200(before EA); 100(after EA)	200 ~ 250	200 ~ 250
Perfusion dose(ml)	5 ml/kg	3 ml/kg	2 ml/kg

EA, electrocardiogram alignment.

The standard for inducing cardiac arrest and reviving

(1) Continuous monitoring indicating a change in blood flow from variation to convection was the criteria for cardioplegic arrest. (2) Electrocardiogram readings indicating the absence of heart rate or a heart rate less than 20 beats per minute. (3) Femoral artery blood pressure dropping below 20 mmHg. Satisfying two out of the three conditions was seen as a sign of cardioplegic arrest¹³.

Continuous monitoring of the femoral artery was necessary to achieve return of spontaneous circulation (ROSC), ensuring a steady and sustained pulsatile waveform while maintaining a pressure of 60 mmHg or higher. Furthermore, the rats showed a regular sinus rhythm on the electrocardiogram recordings.

Blood concentration

The blood was concentrated using a Heraeus 600i high-capacity low-temperature centrifuge during the process. The specified conditions were adjusted to 1000 rotations over a duration of 10 min. After spinning at 1000 g for 1 min, the liquid above the sediment was discarded, and the concentrated hemoglobin in the CPB system was injected into the caudal artery to achieve the target hematocrit level.

Outcomes

The survival of the animals was contingent upon the stability and maintenance of specific physiological indicators within established baseline values. These parameters included a mean arterial pressure (MAP) exceeding 80 mmHg, a heart rate (HR) ranging from 180 to 250 beats per minute, a respiratory rate between 70 and 90 breaths per minute, partial pressure of carbon dioxide in arterial blood (PaCO₂) measuring 35–45 mmHg, partial pressure of oxygen in arterial blood (PaO₂) exceeding 90 mmHg, and a rectal temperature (T) maintained at 35.5–36.5℃. Rats were deemed to have survived and possessed potential for long-term viability if their respiration, blood pressure, and heart rate remained stable for two hours post-weaning from cardiopulmonary bypass (CPB).

Blood gas and biochemical parameter analysis and histology

A femoral artery blood sample was utilized to evaluate the oxygen levels and metabolic condition (EG7+, iStar, Abbott Co. Ltd) at nine specific time intervals: before CPB (T1), at the start of CPB(T2), 10 min after CPB(T3), during cardioplegic arrest(T4), 10 min post-cardioplegic arrest (T5), 20 min post-cardioplegic arrest (T6), during cardiac resuscitation (T7), at the end of CPB (T8), and 2 h after weaning off CPB(T9).The ventricular muscle tissues were taken after 2 h observation. Partial myocardial tissues were collected and then immersed in 4% para-formaldehyde solution for 36 h at a temperature of 4 degrees Celsius. Afterward, the tissues underwent dehydration with a LeicaCM1860 automatic dehydration machine from Germany, followed by embedding in paraffin and sectioning to a thickness of 4µM. After that, the parts were dyed with Hematoxylin and eosin (HE) using an automated staining device (LeicaST5010, Germany). The other myocardial tissue was fixed in glutaraldehyde phosphate solution and placed in refrigerator at 4℃ for 1 week. After rinsed, fixed, dehydrated and soaked, each specimen was sliced with 2 copper mesh sheets. The ultrastructure of myocardial cells was observed under transmission electron microscope.

The standard of histopathological score:

The scoring system includes points for normal (1 point) or disordered (2 points) myocardial structure, absent (1 point), mild (2 points), or severe (3 points) myocardial edema, absent (1 point), mild (2 points), moderate (3 points), or severe (4 points) inflammation, and absent (1 point), scattered cells with ischemic necrosis present (2 points), visible aggregation of ischemic necrosis cells (3 points), or visible dissolved ischemic necrosis cells (4 points) for ischemic necrosis²⁸.

The standard of mitochondrial damage score:

Grade 0 indicated a typical mitochondrial structure with intact particles; Grade I showed a mostly normal structure with some particle loss; Grade II displayed swollen mitochondria with a clear matrix; Grade III exhibited fractured ridges and a solidified matrix; Grade IV demonstrated a total breakdown of membrane integrity, resulting in a vacuolar appearance²⁸.

ELISA arrays

Blood samples were taken from the femoral artery before and 2 h after weaning off CPB. Levels of LDH, LDH-1, CK, and CKMB in the serum were analyzed with an ELISA Kit from Mlbio in Shanghai, China, according to the provided guidelines.

Western blot

Proteomic extraction was accomplished from the left ventricular myocardial tissue. The homogenization process utilized RIPA lysis buffer (supplied by Proteintech, Chicago, USA), augmented with both protease and phosphatase inhibitory substances (sourced from Biosharp, Beijing, China). Centrifugal separation ensued at 12,000 revolutions per minute for a duration of 15 min, after which the supernatant layers were sequestered. Protein measurement was conducted using the BCA Protein Assay Kit manufactured by Solarbio in Beijing, China. Protein concentrations were standardized across samples by carefully adding RIPA lysis buffer. Subsequent to this adjustment, protein fractions were segregated via electrophoretic migration on 10% SDS-polyacrylamide gels, followed by transference onto polyvinylidene fluoride membranes procured from Millipore, Massachusetts, USA. After that, the membranes were blocked using 5% non-fat milk from Nestle in Switzerland for 60 min at room temperature. Following this, the primary antibodies were incubated overnight at 4 °C using Proteintech antibodies that were diluted. Following intermittent rinsing with Tris-buffered saline and Tween 20, membranes were introduced to horseradish peroxidase-linked secondary antibodies (also obtained from Proteintech, Chicago, USA) under room temperature conditions for a span of 1 h. Visual detection of protein ligatures was facilitated by an amplified chemiluminescence detection reagent provided by Beyotime, Shanghai, China, and imaged with the Tanon 5200 Multi detection system (Shanghai, China). Image J software (Version 1.51, National Institutes of Health, USA) was used to perform quantitative band analysis.

Data analysis

The mean ± standard deviation was shown for all values. Statistical analysis was performed using GraphPad Prism 8 software by GraphPad Software Inc. The normality of the data was analyzed using the Shapro-Wilk test, which showed that the data conformed to a normal distribution. Intergroup comparison was done through univariate analysis, and group comparison was conducted using the paired t test. A P-value less than 0.05 was deemed statistically significant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

QBM and FSW participated in the conception and design of the study and were major contributors in writing the manuscript; QBM, WAL, DPC, ZX and WCT established the CPB rat model with a right thoracotomy; WL and DPC collected blood samples and myocardial tissues and sorted out the experimental data; QBM, ZX and WAL performed the statistical analysis. All authors contributed to the interpretation of the results and critically reviewed the first draft. All authors read and approved the final manuscript.

Data availability

The datasets used and analyzed during the current study were available from the first author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.